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A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity.

Gamblin CL, Hardy ÉJ, Chartier FJ, Bisson N, Laprise P - J. Cell Biol. (2014)

Bottom Line: However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined.In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane.The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity.

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Affiliation: Département de Biologie Moléculaire, Biochimie Médicale et Pathologie and Centre de Recherche sur le Cancer, Université Laval, and 2 Axe Oncologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Québec, Québec G1R 3S3, Canada.

ABSTRACT
During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.

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Yrt limits aPKC-dependent apicalization of epithelial cells. (A) Portion of the ventral ectoderm of stage 11 control (da-GAL4) embryos stained for Yrt and aPKC (left) or Dlg and Crb (right). Bar, 10 µm (also applies to B and C). (B and C) Left images illustrate a portion of the ventral ectoderm costained with Flag and aPKC, whereas right images show a costaining of Dlg and Crb in stage 11 embryos expressing Flag-tagged YrtFL (B) or embryos expressing Flag-YrtΔFA (C). (D–G) Ventral ectoderm of stage 13 (St13) embryos (left) or ventral epidermis of stage 16 (St16) embryos (right) costained for Dlg and Crb. The embryonic genotypes were da-GAL4 (D), UAS-aPKCCAAX; da-GAL4 (ubiquitous expression of membrane-targeted aPKC; E), yrt75a (zygotic mutants; F), and UAS-aPKCCAAX; da-GAL4, yrt75a/yrt75a (G). Bar, 20 µm (also applies to E–G). (H) Histogram showing the hatching percentage of control (da-GAL4) embryos, embryos expressing YrtFL, or embryos expressing YrtΔFA. Error bars represent standard deviation. Below the histogram, a Western blot using an anti-Flag antibody shows that Flag-YrtFL and Flag-YrtΔFA are expressed at equivalent levels. Blotting for Actin validated equal loading. Cont., control. (I–L) Cuticle preparation of embryos of the following genotypes: da-GAL4 (I), UAS-aPKCCAAX; da-GAL4 (J), yrt75a (K), and UAS-aPKCCAAX; da-GAL4, yrt75a/yrt75a (L). Bar, 100 µm (also applies to J–L).
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fig4: Yrt limits aPKC-dependent apicalization of epithelial cells. (A) Portion of the ventral ectoderm of stage 11 control (da-GAL4) embryos stained for Yrt and aPKC (left) or Dlg and Crb (right). Bar, 10 µm (also applies to B and C). (B and C) Left images illustrate a portion of the ventral ectoderm costained with Flag and aPKC, whereas right images show a costaining of Dlg and Crb in stage 11 embryos expressing Flag-tagged YrtFL (B) or embryos expressing Flag-YrtΔFA (C). (D–G) Ventral ectoderm of stage 13 (St13) embryos (left) or ventral epidermis of stage 16 (St16) embryos (right) costained for Dlg and Crb. The embryonic genotypes were da-GAL4 (D), UAS-aPKCCAAX; da-GAL4 (ubiquitous expression of membrane-targeted aPKC; E), yrt75a (zygotic mutants; F), and UAS-aPKCCAAX; da-GAL4, yrt75a/yrt75a (G). Bar, 20 µm (also applies to E–G). (H) Histogram showing the hatching percentage of control (da-GAL4) embryos, embryos expressing YrtFL, or embryos expressing YrtΔFA. Error bars represent standard deviation. Below the histogram, a Western blot using an anti-Flag antibody shows that Flag-YrtFL and Flag-YrtΔFA are expressed at equivalent levels. Blotting for Actin validated equal loading. Cont., control. (I–L) Cuticle preparation of embryos of the following genotypes: da-GAL4 (I), UAS-aPKCCAAX; da-GAL4 (J), yrt75a (K), and UAS-aPKCCAAX; da-GAL4, yrt75a/yrt75a (L). Bar, 100 µm (also applies to J–L).

Mentions: To further explore the physiological outcome of the interaction between Yrt and aPKC, we produced a Yrt mutant protein lacking the FA domain that is required for the interaction with aPKC (referred to as YrtΔFA). YrtΔFA was located at both the apical and lateral membranes of ectodermal cells in stage 11 embryos, whereas endogenous Yrt and YrtFL were restricted to the lateral membrane at this stage of embryogenesis (Fig. 4, A–C). This is in agreement with our model that aPKC is responsible for the apical exclusion of Yrt normally prevailing in maturing epidermal cells (Laprise et al., 2006), as suggested by our data with Yrt5A. Thereby, our results provide a molecular basis explaining the spatiotemporal dynamics of Yrt previously described (Laprise et al., 2006). Strikingly, although YrtΔFA is ectopically associated with the apical membrane, it had no impact on epithelial cell polarity in contrast to Yrt5A. Indeed, the apical markers aPKC and Crb as well as the lateral protein Dlg were distributed normally in YrtΔFA-expressing embryos (Fig. 4 C). This implies that apical Yrt impacts on apical–basal polarity at midembryogenesis primarily by binding to aPKC rather than Crb, which also directly associates with Yrt within the apical domain (Laprise et al., 2006). Moreover, expression of YrtΔFA had a limited impact on embryo survival, whereas expression of YrtFL is fully lethal (Fig. 4 H). This further suggests that binding to aPKC is a fundamental function of Yrt and proposes that Yrt controls the action of its negative regulator aPKC. Accordingly, although zygotic expression of aPKCCAAX in wild-type embryos did not interfere with epithelial cell polarity, its expression in a yrt-sensitized background resulted in a striking apicalization phenotype. The latter was characterized by the expansion of Crb expression territories (Fig. 4, D–G, left) followed by extreme extension of the apical membrane of epidermal cells leading to the formation of inverted cysts toward the end of embryogenesis (i.e., apical membrane facing out; Fig. 4 G, right). Consequently, the larval cuticle, secreted through the apical domain, formed small spheres typical of apicalized epidermal cells (Fig. 4, I–L; Wodarz et al., 1995). The aPKCCAAX-induced phenotype in yrt mutant embryos mimics polarity defects associated with a strong Crb gain of function or a loss of the lateral protein Lethal (2) giant larvae (Lgl; Wodarz et al., 1995; Bilder et al., 2000). This is consistent with previous studies showing that aPKC favors apical membrane development by enhancing Crb activity through phosphorylation of its cytoplasmic tail (Sotillos et al., 2004) and via inhibition of Lgl functions by dislodging it from the apical membrane (Plant et al., 2003; Hutterer et al., 2004). These data demonstrate that a reduction of Yrt levels allows for aPKC-dependent apicalization of epithelial cells, thereby highlighting Yrt as a critical inhibitor of aPKC-mediated signaling.


A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity.

Gamblin CL, Hardy ÉJ, Chartier FJ, Bisson N, Laprise P - J. Cell Biol. (2014)

Yrt limits aPKC-dependent apicalization of epithelial cells. (A) Portion of the ventral ectoderm of stage 11 control (da-GAL4) embryos stained for Yrt and aPKC (left) or Dlg and Crb (right). Bar, 10 µm (also applies to B and C). (B and C) Left images illustrate a portion of the ventral ectoderm costained with Flag and aPKC, whereas right images show a costaining of Dlg and Crb in stage 11 embryos expressing Flag-tagged YrtFL (B) or embryos expressing Flag-YrtΔFA (C). (D–G) Ventral ectoderm of stage 13 (St13) embryos (left) or ventral epidermis of stage 16 (St16) embryos (right) costained for Dlg and Crb. The embryonic genotypes were da-GAL4 (D), UAS-aPKCCAAX; da-GAL4 (ubiquitous expression of membrane-targeted aPKC; E), yrt75a (zygotic mutants; F), and UAS-aPKCCAAX; da-GAL4, yrt75a/yrt75a (G). Bar, 20 µm (also applies to E–G). (H) Histogram showing the hatching percentage of control (da-GAL4) embryos, embryos expressing YrtFL, or embryos expressing YrtΔFA. Error bars represent standard deviation. Below the histogram, a Western blot using an anti-Flag antibody shows that Flag-YrtFL and Flag-YrtΔFA are expressed at equivalent levels. Blotting for Actin validated equal loading. Cont., control. (I–L) Cuticle preparation of embryos of the following genotypes: da-GAL4 (I), UAS-aPKCCAAX; da-GAL4 (J), yrt75a (K), and UAS-aPKCCAAX; da-GAL4, yrt75a/yrt75a (L). Bar, 100 µm (also applies to J–L).
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fig4: Yrt limits aPKC-dependent apicalization of epithelial cells. (A) Portion of the ventral ectoderm of stage 11 control (da-GAL4) embryos stained for Yrt and aPKC (left) or Dlg and Crb (right). Bar, 10 µm (also applies to B and C). (B and C) Left images illustrate a portion of the ventral ectoderm costained with Flag and aPKC, whereas right images show a costaining of Dlg and Crb in stage 11 embryos expressing Flag-tagged YrtFL (B) or embryos expressing Flag-YrtΔFA (C). (D–G) Ventral ectoderm of stage 13 (St13) embryos (left) or ventral epidermis of stage 16 (St16) embryos (right) costained for Dlg and Crb. The embryonic genotypes were da-GAL4 (D), UAS-aPKCCAAX; da-GAL4 (ubiquitous expression of membrane-targeted aPKC; E), yrt75a (zygotic mutants; F), and UAS-aPKCCAAX; da-GAL4, yrt75a/yrt75a (G). Bar, 20 µm (also applies to E–G). (H) Histogram showing the hatching percentage of control (da-GAL4) embryos, embryos expressing YrtFL, or embryos expressing YrtΔFA. Error bars represent standard deviation. Below the histogram, a Western blot using an anti-Flag antibody shows that Flag-YrtFL and Flag-YrtΔFA are expressed at equivalent levels. Blotting for Actin validated equal loading. Cont., control. (I–L) Cuticle preparation of embryos of the following genotypes: da-GAL4 (I), UAS-aPKCCAAX; da-GAL4 (J), yrt75a (K), and UAS-aPKCCAAX; da-GAL4, yrt75a/yrt75a (L). Bar, 100 µm (also applies to J–L).
Mentions: To further explore the physiological outcome of the interaction between Yrt and aPKC, we produced a Yrt mutant protein lacking the FA domain that is required for the interaction with aPKC (referred to as YrtΔFA). YrtΔFA was located at both the apical and lateral membranes of ectodermal cells in stage 11 embryos, whereas endogenous Yrt and YrtFL were restricted to the lateral membrane at this stage of embryogenesis (Fig. 4, A–C). This is in agreement with our model that aPKC is responsible for the apical exclusion of Yrt normally prevailing in maturing epidermal cells (Laprise et al., 2006), as suggested by our data with Yrt5A. Thereby, our results provide a molecular basis explaining the spatiotemporal dynamics of Yrt previously described (Laprise et al., 2006). Strikingly, although YrtΔFA is ectopically associated with the apical membrane, it had no impact on epithelial cell polarity in contrast to Yrt5A. Indeed, the apical markers aPKC and Crb as well as the lateral protein Dlg were distributed normally in YrtΔFA-expressing embryos (Fig. 4 C). This implies that apical Yrt impacts on apical–basal polarity at midembryogenesis primarily by binding to aPKC rather than Crb, which also directly associates with Yrt within the apical domain (Laprise et al., 2006). Moreover, expression of YrtΔFA had a limited impact on embryo survival, whereas expression of YrtFL is fully lethal (Fig. 4 H). This further suggests that binding to aPKC is a fundamental function of Yrt and proposes that Yrt controls the action of its negative regulator aPKC. Accordingly, although zygotic expression of aPKCCAAX in wild-type embryos did not interfere with epithelial cell polarity, its expression in a yrt-sensitized background resulted in a striking apicalization phenotype. The latter was characterized by the expansion of Crb expression territories (Fig. 4, D–G, left) followed by extreme extension of the apical membrane of epidermal cells leading to the formation of inverted cysts toward the end of embryogenesis (i.e., apical membrane facing out; Fig. 4 G, right). Consequently, the larval cuticle, secreted through the apical domain, formed small spheres typical of apicalized epidermal cells (Fig. 4, I–L; Wodarz et al., 1995). The aPKCCAAX-induced phenotype in yrt mutant embryos mimics polarity defects associated with a strong Crb gain of function or a loss of the lateral protein Lethal (2) giant larvae (Lgl; Wodarz et al., 1995; Bilder et al., 2000). This is consistent with previous studies showing that aPKC favors apical membrane development by enhancing Crb activity through phosphorylation of its cytoplasmic tail (Sotillos et al., 2004) and via inhibition of Lgl functions by dislodging it from the apical membrane (Plant et al., 2003; Hutterer et al., 2004). These data demonstrate that a reduction of Yrt levels allows for aPKC-dependent apicalization of epithelial cells, thereby highlighting Yrt as a critical inhibitor of aPKC-mediated signaling.

Bottom Line: However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined.In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane.The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Biologie Moléculaire, Biochimie Médicale et Pathologie and Centre de Recherche sur le Cancer, Université Laval, and 2 Axe Oncologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Québec, Québec G1R 3S3, Canada.

ABSTRACT
During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.

Show MeSH